The visceral epithelial cell (podocyte) is a fundamental component of the capillary wall of the renal glomerulus, contributing to its structural stability, as well as regulating fluid filtration and assuring protein impermeability. Loss of podocytes from the glomerulus is a pathologic hallmark of the progression of diabetic nephropathy. Podocytes can be recovered in the urine (podocyturia). Since podocytes do not replicate post-natally, quantitative podocyturia represents a non-invasive index of ongoing glomerular podocyte losses. However, current methods for quantifying podocyturia are too labor-intensive to be applied to large-scale intervention trials for the prevention of diabetic nephropathy. This proposal brings together groups with expertise in diabetic nephropathy and the cell biology of the podocyte and in applications of FACS technology to develop a more cost-effective, rapid and flexible methodology for quantifying podocyturia. Following enrichment of the very low density of podocytes present in the urine and staining with fluorescent monoclonal antibodies to multiple podocyte-specific markers, automated counting using an 11-color (Hi-D) FACS will determine the numbers of viable, non-viable and apoptotic podocytes in the sample. The Hi-D FACS will allow gating out of the carrier cells needed to increase the recovery of podocytes from the urine. After its development, the FACS-based method will be applied to a longitudinal study of patients with type 1 diabetes both with and without evidence of nephropathy. The specific hypotheses to be tested in the R33 phase are 1) does the time course of development of abnormal podocyturia in patients with no abnormalities at baseline precede that of microalbuminuria, i.e. is podocyturia an earlier biomarker of incipient nephropathy? and 2) do the responses of albuminuria and podocyturia to treatment with angiotensin-converting enzyme inhibitors differ?